Hepatitis C virus (HCV) causes chronic liver infections, often leading to cirrhosis and carcinoma. Understanding certain aspects of its life cycle is complicated by the failure of HCV to replicate efficiently in cell culture. In particular, little is known about the mechanism of assembly of HCV virions. HCV is evolutionarily closely related to pestiviruses, which grow efficiently in cell cultures. We have identified mutant pestiviruses with a novel defect in assembly that reveals an unprecedented role of nonstructural proteins in virion asssembly. We hypothesize that nonstructural proteins play a role in pestivirus and hepacivirus assembly. The research proposed here will exploit a pestivirus system as a model to analyze the mechanisms virion assembly and the essential role of non-structural proteins in this process. We will utilize genetic, biochemical and structural approaches to identify and characterize the role of nonstructural proteins in virion assembly. Specifically we propose to: 1. Map critical sites of the pestivirus nonstructural polyprotein involved in virion assembly. 2. Identify and characterize pestivirus proteins involved in the assembly process using a proteomic approach. 3. Characterize the subcellutar localization of pestivirus assembly sites involving nonstructural proteins. 4. Screen the pestivirus nonstructural polyprotein to identify additional loci involved in virion assembly. This research will expand our knowledge on the novel role of nonstructural proteins in pestivirus assembly. This information may help delineate the assembly of HCV when cell culture systems for efficient HCV replication become available.